The emergence of resistant bacterial strains without the increased development of new antibiotic structure classes constitutes a serious medical crisis. Brown, E. D.; Wright, G. D. Chem. Rev. 2005, 105, 759-774; Coates, A.; Hu, Y.; Bax, R.; Page, C. Nat. Rev. Drug Discovery 2002, 1, 895-910. Infection with the common pathogen Staphylococcus aureus has been estimated to double the cost, length of stay, and the even death rate in New York City hospitals. Rubin, R. J.; Harrington, C. A.; Poon, A.; Dietrich, K.; Greene, J. A.; Moiduddin, A. Emerging Infectious Diseases 1999, 5, 9-17. Designing antibiotics that treat bacterial infections is a constant struggle for synthetic chemists and biologists because bacteria have an extraordinary ability to adapt and develop resistance to new antibacterial agents. For example, the most recent antibiotic, Linezolid, was released on the market in 2000, only to have cases of Linezolid-resistant bacteria reported the following year. This was alarming news, because Linezolid is a member of the oxazolidinone family, a structure class that had never previously been used as a scaffold for antibacterial agents. This development underscores the need for the discovery of new structural scaffolds with antibacterial activity.
Combinatorial chemistry continues to play an important role in advancing the chemical biology and drug discovery fields. Navre, M., Application of combinatorial chemistry to antimicrobial drug discovery. Expert Opin. Invest. Drugs 1998, 7, 1257-1269; Seneci, P.; Miertus, S., Combinatorial chemistry and high-throughput screening in drug discovery: Different strategies and formats. Mol. Diversity. 2000, 5, 75-89. One of the main advantages of combinatorial chemistry is the ability to generate a large, diverse library of compounds using a minimum amount of reagents in a relatively short amount of time. Because a combinatorial approach can generate a large number of compounds, this makes it ideal for probing and studying biological targets.
Solid-phase chemistry has taken on a major role in advancing combinatorial chemistry. Ganesan, A., Recent developments in combinatorial organic synthesis. Drug Discovery Today 2002, 7, 47-55; Balasubramanian, S., Solid phase chemical technologies for combinatorial chemistry. J. Cell. Biochem. 2001, 28-33; Bannwarth, W., Solid phase chemistry. Linkers for solid-phase organic synthesis (SPOS) and combinatorial approaches on solid support. Methods Princ. Med. Chem. 2000, 9, 47-98. Traditional solid phase techniques employ hydrophobic polymeric supports, such as polystyrene beads. Yu, Z. R.; Bradley, M., Solid supports for combinatorial chemistry. Curr. Opin. Chem. Biol. 2002, 6, 347-352. Although these solid supports offer advantages, including rapid and easy compound purification, there are some disadvantages. The hydrophobic nature of polystyrene beads is not compatible with many reactions that require the use of aqueous or certain polar solvents. Recently, the implementation of small molecule macroarrays in combinatorial chemistry has lead to an improved ability to perform both on- and off-support biological assays. Blackwell, H. E., Hitting the SPOT: small-molecule macroarrays advance combinatorial synthesis. Curr. Opin. Chem. Biol. 2006, 10, 203-212; Bowman, M. D.; Jacobson, M. M.; Blackwell, H. E., Discovery of fluorescent cyanopyridine and deazalumazine dyes using small molecule macroarrays. Org. Lett. 2006, 8, 1645-1648; Bowman, M. D.; Jacobson, M. M.; Pujanauski, B. G.; Blackwell, H. E., Efficient synthesis of small molecule macroarrays: optimization of the macroarray synthesis platform and examination of microwave and conventional heating methods. Tetrahedron 2006, 62, 4715-4727; Lin, Q.; Blackwell, H. E., Rapid synthesis of diketopiperazine macroarrays via Ugi four-component reactions on planar solid supports. Chem. Commun. 2006, 2884-2886.
Solid phase synthesis requires a linker to attach or “link” a synthesized substrate to an insoluble support. A variety of linkers have been used in solid phase synthesis, with two of the most widely used being the Wang and Rink linkers. James, I. W., Linkers for solid phase organic synthesis. Tetrahedron 1999, 55, 4855-4946. These two acid labile linkers are advantageous for synthesis because they can be cleaved with relatively mild acids in a short period of time.
Small molecule macroarrays can be traced back to the origins of the SPOT-synthesis technique. Frank, R., Spot-Synthesis—an Easy Technique for the Positionally Addressable, Parallel Chemical Synthesis on a Membrane Support. Tetrahedron 1992, 48, 9217-9232. Frank originally designed the SPOT-synthesis technique for the construction of peptide libraries as an alternative to standard solid phase peptide synthesis approaches (i.e. the use of polystyrene beads). Using the SPOT technique individual polypeptides can be synthesized in a spatially addressed format, and the resulting polypeptide arrays can be used in a variety of on support biological assays.
The generation of small molecule macroarrays involves the use of a planar cellulose support for library construction. This cellulose support is readily accessible laboratory filter paper, an inexpensive alternative to other solid-phase supports. A variety of organic compounds can be used as building blocks for constructing arrays of small molecules. Recently, Blackwell et al. has constructed small molecule macroarrays utilizing multi-component reactions, and microwave irradiation to construct libraries of heterocylces, chalcones, diketopiperazines, and fluorescent cyanopyridine and deazalumazine dyes. Bowman, M. D.; Jeske, R. C.; Blackwell, H. E., Microwave-accelerated SPOT-synthesis on cellulose supports. Org. Lett. 2004, 6, 2019-2022; Lin, Q.; O'Neill, J. C.; Blackwell, H. E., Small molecule macroarray construction via Ugi four-component reactions. Org. Lett. 2005, 7, 4455-4458; Bowman, M. D.; Jacobson, M. M.; Blackwell, H. E., Discovery of fluorescent cyanopyridine and deazalumazine dyes using small molecule macroarrays. Org. Lett. 2006, 8, 1645-1648; Bowman, M. D.; Jacobson, M. M.; Pujanauski, B. G.; Blackwell, H. E., Efficient synthesis of small molecule macroarrays: optimization of the macroarray synthesis platform and examination of microwave and conventional heating methods. Tetrahedron 2006, 62, 4715-4727. Small molecule macroarrays have advantages over traditional solution-phase synthesis, as several hundred compounds can be synthesized in high purity and screened for biological activity in a few days using a minimal amount of reagents, for example as illustrated in FIG. 1.
Application of a combinatorial approach to the identification of antibacterial agents permits the generation of diverse arrays of compounds that can be screened for antibacterial activity. Several new antibacterial agents have been identified in combinatorial libraries using a variety of screening techniques, including pyrrolidine bis-cyclic guanidines, hydrazinyl urea-based compounds, benzopyrans, thymidinyl derivatives, and natural product derivatives, and certain 1,3-diphenyl-2-propen-1-ones (chalcones). Hensler, M. E.; Bernstein, G.; Nizet, V.; Nefzi, A., Pyrrolidine bis-cyclic guanidines with antimicrobial activity against drug-resistant Gram-positive pathogens identified from a mixture-based combinatorial library. Bioorg. Med. Chem. Lett. 2006, 16, 5073-5079; Nicolaou, K. C.; Roecker, A. J.; Barluenga, S.; Pfefferkorn, J. A.; Cao, G. Q., Discovery of novel antibacterial agents active against methicillin-resistant Staphylococcus aureus from combinatorial benzopyran libraries. Chembiochem 2001, 2, 460-465; Sun, D.; Lee, R. E., Solid-phase synthesis development of a thymidinyl and 2′-deoxyuridinyl Ugi library for anti-bacterial agent screening. Tetrahedron Lett. 2005, 46, 8497-8501; Shi, S.; Zhu, S.; Gerritz, S. W.; Esposito, K.; Padmanabha, R.; Li, W.; Herbst, J. J.; Wong, H.; Shu, Y. Z.; Lam, K. S.; Sofia, M. J., Solid-phase synthesis and anti-infective activity of a combinatorial library based on the natural product anisomycin. Bioorg. Med. Chem. Lett. 2005, 15, 4151-4154; Ansari, F. L.; Nazir, S.; Noureen, H.; Mirza, B., Combinatorial synthesis and antibacterial evaluation of an indexed chalcone library. Chem. Biodiv. 2005, 2, 1656-1664.
Chalcones are small molecule natural products found in a variety of plants that exhibit a wide range of biological activities. Kromann, H.; Larsen, M.; Boesen, T.; Schonning, K.; Nielsen, S. F., Synthesis of prenylated benzaldehydes and their use in the synthesis of analogues of licochalcone A. Eur. J. Med. Chem. 2004, 39, 993-1000; Jun, N.; Hong, G.; Jun, K., Synthesis and evaluation of 2′,4′,6′-trihydroxychalcones as a new class of tyrosinase inhibitors. Bioorg. Med. Chem. 2007, 15, 2396-2402; Lawrence, N. J.; Patterson, R. P.; Ooi, L.-L.; Cook, D.; Ducki, S., Effects of a-substitutions on structure and biological activity of anticancer chalcones. Bioorg. Med. Chem. Lett. 2006, 16, 5844-5848; Modzelewska, A.; Pettit, C.; Achanta, G.; Davidson, N. E.; Huang, P.; Khan, S. R., Anticancer activities of novel chalcone and bis-chalcone derivatives. Bioorg. Med. Chem. 2006, 14, 3491-3495.
Certain chalcones exhibit antimicrobial activity. Sivakumar, P. M.; Seenivasan, S. P.; Kumar, V.; Doble, M., Synthesis, antimycobacterial activity evaluation, and QSAR studies of chalcone derivatives. Bioorg. Med. Chem. Lett. 2007, 17, 1695-1700; Gafner, S.; Wolfender, J.-L.; Mavi, S.; Hostettmann, K., Antifungal and antibacterial chalcones from Myrica serratia. Planta Med. 1996, 62, 67-9. Naturally-occurring chalcones (shown below) are generally lipophilic and have moderate antibacterial activity. There have been solution-phase synthetic efforts directed at improving the antibacterial activity of naturally-occurring chalcones by increasing their water solubility. Nielsen, S. F.; Boesen, T.; Larsen, M.; Schonning, K.; Kromann, H., Antibacterial chalcones-bioisosteric replacement of the 4′-hydroxy group. Bioorg. Med. Chem. 2004, 12, 3047-3054; Nielsen, S. F.; Larsen, M.; Boesen, T.; Schonning, K.; Kromann, H., Cationic chalcone antibiotics. design, synthesis, and mechanism of action. J. Med. Chem. 2005, 48, 2667-2677.

Chalcones should thus be a useful scaffold for making and assessing small molecules for antimicrobial activity. Furthermore, chalcones are adaptable to macroarray methods due to their relatively straightforward synthesis. The key feature of combinatorial chemistry—the speed at which a large number of diverse compounds can be generated—can be applied to the rapid discovery of new lead structures for use as antibacterial agents. The generation of small molecule macroarrays can streamline the process for generating diverse small molecule libraries with potential antibacterial activities, and can be used to identify novel antimicrobial agents, including antibacterial agents.
Backwell et al. WO 2008/016738 (published Feb. 7, 2008) have reported making chalcone-based small molecule macroarrays including chalcones, and cyanopyridine and methylpyrimidine derivatives of chalcones and the screening of the compound libraries made for antibacterial activity. These macroarrays employed planar cellulose membranes derivatized with a Wang-type linker. See: Bowman, et al. Tetrahedron 2006.
Bacterial cellular membranes have been identified as a possible target of antibacterial agents. Bacterial membranes are composed mostly of negatively charged phospholipid, phosphatidylglycerol. In contrast, eukaryotic cellular membranes comprise two different phospholipids, phosphatidylcholine and sphingomyelin. Zasloff, M., Antimicrobial peptides of multicellular organisms. Nature 2002, 415, 389-395. The differences in the composition of bacterial and eukaryotic membranes represent a unique structural difference that may be exploited as an antibacterial target. This is shown in the effectiveness of certain antimicrobial peptides, which are inherently present in humans, termed host-defense peptides. Host-defense peptides are short peptides (12-50 amino acids) that are found in a variety of living organisms including humans, and there have been synthetic examples of mimicking host-defense peptides for use as a potential antibacterial therapeutic. Schmitt, M. A.; Weisblum, B.; Gellman, S. H., Unexpected relationships between structure and function in alpha-, beta-peptides: antimicrobial foldamers with heterogeneous backbones. J. Am. Chem. Soc. 2004, 126, 6848-6849; Epand, R. F.; Raguse, T. L.; Gellman, S. H.; Epand, R. M., Antimicrobial 14-Helical beta-Peptides: Potent Bilayer Disrupting Agents. Biochemistry 2004, 43, 9527-9535; Schmitt, M. A.; Weisblum, B.; Gellman, S. H., Interplay among Folding, Sequence, and Lipophilicity in the Antibacterial and Hemolytic Activities of alpha/beta-Peptides. J. Am. Chem. Soc. 2007, 129, 417-428. Most of these amphipathic peptides contain structural features that are believed to contribute to their antibacterial activity, including regions of positively charged amino acid residues (for attraction to negatively charged bacterial membranes), and regions of hydrophobic amino acid residues (for insertion and subsequent disruption of the membrane).
Peptoids, or N-substituted glycine oligomers, are possible alternatives to antimicrobial peptides because they are resistant to proteolytic degradation and diverse libraries with a variety of sidechains can be generated using commercially available amines.
The present invention relates to additional methods for synthesis of small molecule macroarrays of chalcones and derivatives thereof and screening of such arrays for useful biological activities, including therapeutic activities and particularly antimicrobial activities. The invention relates in a second aspect to methods for covalently linking amino acids, peptides and/or peptoids to the chalcones and chalcone derivatives of such macroarrays to expand the potential for new antimicrobial compounds. The invention additionally relates to novel chalcones and chalcone derivatives exhibiting antimicrobial, particularly antibacterial activity.